Starch granules containing a recombinant polypeptide of interest, method for obtaining them, and their uses

ABSTRACT

The present invention relates to starch granules containing a fusion polypeptide between a starch synthase and a recombinant polypeptide of interest, the nucleotide sequences used for obtaining them, their methods of preparation, as well as their uses, especially in pharmaceutical compositions.

This application is a 371 of PCT/FR00/01384 filed May 19, 2000.

The present invention relates to starch granules containing arecombinant polypeptide of interest, a method of obtaining them, as wellas their uses, especially in pharmaceutical compositions.

Starch is one of the world's most important sources of polysaccharides,occurring in particular in plants (maize, potato, wheat, rice, barley,etc.), algae, micro-algae etc.

Starch occurs in the form of granules that are insoluble in water, thesize of which can vary from 0.1 to several tens of μm in diameterdepending on its origin (plants, algae or micro-algae) or even thegenotype of the plant in question. Thus, the sizes of these granulesvary from 0.1 μm in diameter to more than 50 μm in diameter.Furthermore, the degrees of crystallinity of these granules range from0% (for granules rich in amylose) to over 30%. There are three or fourcrystalline types (A, B, C, V). The granule grows by the laying-down ofalternately amorphous and semicrystalline layers starting from thecentre of the starch granule.

Starch contains several distinct polysaccharide fractions, composed ofglucans bound at α-1,4 and branched at α-1,6. More particularly, starchconsists of two glucose polymers: amylose on the one hand, the minorfraction of the granule (about 20–30 wt. %), of low molecular weight,with little branching (<1% of α-1,6 bonds) and amylopectin on the otherhand, the major fraction of the granule (70–80 wt. %), of high molecularweight and highly branched (5% of α-1,6 bonds). Amylose is not necessaryfor the development of crystallinity of the starch granule; it is nowknown that it is amylopectin that is responsible for the crystallinityof the starch granule.

In biological terms, strictly speaking starch only occurs in the plantkingdom, and more specifically in the chloroplasts or in thenon-photosynthetic granules of the eukaryotic plant cell. Two types ofstarch can be synthesized by plants: temporary or photosynthetic starch(synthesis of which takes place at the level of the chloroplasts), andreserve starch (synthesis of which takes place at the level of theamyloplasts).

Synthesis of starch in plants involves a whole panoply of enzymes takingpart in biosynthesis of the precursor ADP-glucose, scaffolding of theamylose and amylopectin molecules and, finally, degradation of thestarch granule.

The first stage in the biosynthesis of starch is the production of theprecursor ADP-glucose with the involvement of the two enzymes:phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase).

The second stage in the biosynthesis of the starch granule also involvestwo types of enzymes, mainly taking part in the synthesis of amylose andamylopectin: starch synthases (or adenosine diphosphate glucoseα-1,4-glucan α-4-glucosyltransferases) and branching enzymes (orα-1,4-glucan-6-glucosyltransferases). The starch synthases catalyse thetransfer of the glucose residue from ADP-glucose onto growing chains ofglucans by creating an O-glycosidic bond of type α-1,4. Then thebranching enzymes hydrolyse an α-1,4 bond of an elongating glucan, andthen join the fragment thus released onto the remainder of the glucan bymeans of an α-1,6 bond.

With regard to starch degradation, there are two main families ofdegrading enzymes: the hydrolytic enzymes (hydrolases) on the one hand,such as α-amylases (endomylases), β-amylases (exomylases), γ-amylases(amyloglucosidases), D-enzymes (glucosyltransferases), R-enzymes(debranching enzymes), α-glucosidases (maltases) and, on the other hand,the phosphorolytic enzymes (or starch phosphorylases).

Several isoforms of starch synthases occur together in higher plants.The main difference between these isoforms relates to their solubility(i.e. they are dissolved in the plastid stroma in plants) or to the factthat they are bound to the starch granule.

The starch synthases bound to the starch granule (or GBSS: Granule BoundStarch Synthases) occur in close association with starch. Severalisoforms of GBSS have been isolated in maize, pea, potato or wheat(MacDonald and Preiss, 1985; Smith, 1990; Dry et al., 1992; Denyer etal., 1995). In all cases, GBSSI is the main isoform; the part played bythis isoform in the biogenesis of the starch granule is the formation ofamylose (Tsai, 1974; Hovenkamp-Hermelink et al., 1987; Delrue et al.,1992; Denyer et al., 1995). A mutation at the loci WX of cereals, AMF ofthe potato, and LAM of the pea, combines disappearance of GBSSI withcomplete collapse of the amylose fraction of starch. A cDNAcorresponding to the “Waxy protein” (through a misuse of language, theterm “Waxy protein” is employed to designate the GBSSI in plants, thusdistinguishing it from other GBSS) has been isolated in wheat, barley,maize, rice, potato and pea. Comparisons of the relative proteinsequences show there is considerable homology between the differentspecies (Ainsworth et al., 1993).

GBSSI is not the only starch synthase bound to the starch granule. Otherisoforms are found bound to the starch granule in pea, potato, maize orwheat (Smith, 1990; Dry et al., 1992; Mu et al, 1994; Denyer et al,1995). However, the roles of these various isoforms are not yet clear.Furthermore, most of them are also found in the soluble phase.

The soluble starch synthases (SS) are not bound to the starch granule,but are found in soluble form in the plastid stroma of plants. As withthe bound forms, several forms of soluble starch synthases occur in thehigher plants. For example, three isoforms of soluble starch synthases(SSI, SSII and SSIII) have been detected in the potato tuber.

cDNA's corresponding to various forms of soluble starch synthases havebeen cloned in higher plants (Baba et al, 1993; Dry et al, 1992; Edwardset al, 1995; Abel et al, 1996; Marshall et al, 1996; Gao et al, 1998).Sequence comparison flowing from this clearly shows the presence ofthree regions that are highly conserved across the isoforms, whetherwithin a single species or between species of higher plants.

Recent research by the Inventors made it possible to establish thatsoluble starch synthase II (SSII) from Chlamydomonas reinhardtii ismainly involved in the formation of crystals of the amylopectinmolecule.

On the other hand, GBSSI does not take part in the construction ofamylopectin crystals. GBSSI activity has never been detected in thesoluble phase. GBSSI is intimately associated with the starch granule.However, in contrast to amylase, no unit binding to starch has beenfound in the GBSSI sequences described so far. Accordingly, themechanism controlling the binding of GBSSI to the starch granule isunknown.

Starch synthases are of particular interest in that these enzymes mightmake it possible to transport a recombinant peptide of interest towardsthe plastids where the biosynthesis of starch granules takes place.Thus, the transformation of plants with sequences coding for fusionpeptides between a starch synthase and a peptide of interest would makeit possible to obtain starch granules in large quantity, from which thesaid peptide of interest could be recovered.

It was with this objective that the authors of International ApplicationWO 98/14601 (Exseed Genetics) described nucleotide sequences coding forfusion proteins in which the polypeptide of interest is bound to theamino terminal end of a starch synthase selected from the groupcomprising soluble starch synthases I, II and III (SSI, SSII, SSIII),granule bound starch synthases (GBSS), branching enzymes I, Ia and IIBband the glucoamylases. However, no method of transformation of plants bymeans of the sequences described in that application, and hence ofobtaining starch granules transformed by the said sequences, isillustrated in detail.

The present invention arises from the demonstration by the inventorsthat only the transformation of plants with nucleotide sequences codingfor fusion polypeptides in which the polypeptide of interest is bound tothe carboxy terminal end of the starch synthase makes it possible toobtain starch granules containing the said peptide of interest.

One of the aims of the present invention is to provide novel nucleotidesequences coding for fusion proteins capable of transporting a peptideof interest towards the site of biosynthesis of the starch granules inplant cells (including the cells of algae or micro-algae).

Another aim of the present invention is to provide plants that have beentransformed by means of the aforementioned nucleotide sequences, thesaid plants producing starch containing a polypeptide of interest.

Another aim of the present invention is to provide starch granulescontaining a polypeptide of interest.

Another aim of the present invention is to provide a method ofpreparation of these starch granules.

Another aim of the present invention is to provide a method ofpreparation of a recombinant polypeptide of interest starting from thesestarch granules.

Another aim of the present invention is to provide compositions,especially pharmaceutical or for foodstuffs, containing theaforementioned starch granules.

Another aim of the present invention is to provide a method ofbiotransformation of starch granules when the said peptide of interestthat is used is capable of transforming starch.

The invention will be illustrated below with the aid of the followingdiagrams:

FIG. 1: cDNA coding for the carboxy terminal part of the GBSSI ofChlamydomonas reinhardtii (SEQ ID NOS 10–12 respectively in order ofappearance).

FIG. 2: cDNA coding for the GBSSI of Chlamydomonas reinhardtii, andpeptide sequence of the GBSSI of Chlamydomonas reinhardtii (SEQ ID NOS1, 3 and 15, respectively in order of appearance).

The present invention relates to any recombinant nucleotide sequencecharacterized in that it comprises, in the direction 5′→3′, a nucleotidesequence coding for an adenosine diphosphate glucose α-1,4-glucanα-4-glucosyltransferase or starch synthase (EC 2.4.1.21), or for aprotein derived from this enzyme, especially by suppression, addition orsubstitution of one or more amino acids, the said starch synthase orderived protein having the property of migrating to the sites ofbiosynthesis of starch granules in plant cells and of attaching to thestarch granules, the said nucleotide sequence coding for the enzyme oraforementioned protein being positioned upstream of a nucleotidesequence coding for a peptide or polypeptide of interest.

By starch synthase we mean, in the foregoing and hereinafter, anyprotein having the property of migrating to the sites of biosynthesis ofstarch granules in plant cells and of attaching to the starch granules,whether or not this starch synthase has conserved its enzymatic activitywithin the fusion polypeptide, encoded by an aforementioned recombinantnucleotide sequence, between the said starch synthase and the saidpolypeptide of interest.

Preferably, the nucleotide sequence coding for a starch synthase, or fora derived protein as defined above, is selected from those coding for astarch synthase bound to the starch granule GBSS that occurs inparticular in plants, algae or micro-algae, and even more advantageouslyfor an isoform GBSSI, or for a protein derived from this GBSS, or GBSSI,as defined above.

The invention relates more particularly to any recombinant nucleotidesequence as defined above, characterized in that the nucleotide sequencecoding for a starch synthase, and more particularly for a GBSS, andespecially for a GBSSI, is such as is obtained by screening a cDNAlibrary prepared from cells that are likely to contain this enzyme,especially from cells of plants, algae or micro-algae, by means of anantiserum containing antibodies specifically recognizing the said starchsynthase coded by one or more cDNA in the library, when the said starchsynthase is expressed by a suitable cloning vector, the said antiserumbeing obtained by immunization of an animal, such as a rabbit, withstarch extracted from the aforementioned cells.

The invention relates more particularly to any recombinant nucleotidesequence as defined above, characterized in that the nucleotide sequencecoding for a starch synthase, or for a derived protein, is selectedfrom:

-   -   the nucleotide sequence of the cDNA of about 2900 to 3100 base        pairs, and of which the 1696 base pairs of the 3′ end are shown        in FIG. 1, the said nucleotide sequence:        -   coding for the GBSSI of Chlamydomonas reinhardtii of about            640 to 680 amino acids, especially of about 660 amino acids,            of which the amino terminal end corresponds to the following            succession of amino acids: ALDIVMVAAEVAPGGKTGGLGDV (SEQ ID            NO: 13), or ALDIVMVAAEVAPWSKTGGLGDV (SEQ ID NO:14), and of            which the carboxy terminal end corresponds to the succession            of amino acids shown in FIG. 1,        -   and being obtained by screening a cDNA library prepared from            cells of Chlamydomonas reinhardtii, by means of an antiserum            obtained by immunization of rabbits with the starch            extracted from the aforementioned cells of Chlamydomonas            reinhardtii,    -   or a nucleotide fragment of the aforementioned cDNA, coding for        a peptide fragment of the GBSSI of Chlamydomonas reinhardtii,        the said peptide fragment comprising the whole of the amino        terminal part of the said GBSSI, and being delimited at its        carboxy terminal end by the amino acid located in one of the        positions 25 to 238, or in one of the positions 118 to 238, of        the amino acid sequence shown in FIG. 1,    -   or a nucleotide sequence derived by degeneration of the genetic        code of the nucleotide sequence of the aforementioned cDNA, or        of an aforementioned nucleotide fragment of the latter, and        coding for the aforementioned GBSSI of Chlamydomonas        reinhardtii, or for an aforementioned peptide fragment of the        latter,    -   or a nucleotide sequence derived from an aforementioned        nucleotide sequence or fragment, especially by substitution,        suppression or addition of one or more nucleotides, and encoding        a peptide sequence derived from the aforementioned GBSSI of        Chlamydomonas reinhardtii, or derived from an aforementioned        peptide fragment of the latter, and having the property of        attaching to the starch granules, the said derived nucleotide        sequence preferably having a homology of at least about 50%, and        preferably of at least about 70%, with the aforementioned        nucleotide sequence or fragment,    -   or a nucleotide sequence capable of hybridization with one of        the aforementioned nucleotide sequences or fragments, especially        in the strict conditions of hybridization defined later,    -   the property possessed by a starch synthase, or a fragment or a        protein derived from the latter as defined above, of being able        to attach to the starch granules, being measurable by the        following technique: extraction of the proteins from the starch        granules, for example according to the method described in        detail below, and detection of the presence of the said starch        synthase, or of a fragment or of a protein derived from the        latter as defined above, especially by polyacrylamide gel        electrophoresis according to the technique described in detail        later.

The invention relates more particularly to any recombinant nucleotidesequence as defined above, characterized in that the aforementionednucleotide sequence coding for a starch synthase, or for a derivedprotein, is more particularly selected from:

-   -   the nucleotide sequence of cDNA shown in FIG. 2, corresponding        to SEQ ID NO:1 in the sequence list given later, the said        nucleotide sequence coding for the GBSSI of Chlamydomonas        reinhardtii,    -   any fragment as defined above of the nucleotide sequence SEQ ID        NO:1 shown in FIG. 2, and more particularly any sequence of        which the nucleotide of the 5′ end corresponds to that located        in one of the positions 1 to 186 of SEQ ID NO:1, and of which        the nucleotide of the 3′ end corresponds to that located in one        of the positions 1499 to 3117 of SEQ ID NO: 1, especially:        -   the sequence SEQ ID NO: 2 delimited by the nucleotides            located in positions 15 to 2138 of SEQ ID NO:1, coding for            the GBSSI of Chlamydomonas reinhardtii in the form of            pre-protein of 708 amino acids (SEQ ID NO: 3) delimited by            the amino acids located at positions 1 and 708 of the            peptide sequence shown in FIG. 2,        -   the sequence SEQ ID NO: 4 delimited by the nucleotides            located at positions 186 to 2138 of SEQ ID NO: 1, coding for            the GBSSI of Chlamydomonas reinhardtii in the form of a            mature protein of 651 amino acids (SEQ ID NO: 5) delimited            by the amino acids located at positions 58 and 708 of the            peptide sequence shown in FIG. 2,        -   the sequence SEQ ID NO: 6 delimited by the nucleotides            located at positions 186 to 1499 of SEQ ID NO: 1, coding for            a fragment of 438 amino acids (SEQ ID NO: 7) delimited by            the amino acids located at positions 58 and 495 of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,        -   the sequence SEQ ID NO: 8 delimited by the nucleotides            located at positions 186 to 1778 of SEQ ID NO:1, coding for            a fragment of 531 amino acids (SEQ ID NO: 9) delimited by            the amino acids located at positions 58 and 588 of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,    -   or a nucleotide sequence derived by degeneration of the genetic        code of the aforementioned nucleotide sequences, and coding for        the aforementioned GBSSI of Chlamydomonas reinhardtii, or for an        aforementioned peptide fragment of the latter,    -   or a nucleotide sequence derived from an aforementioned        nucleotide sequence or fragment, especially by substitution,        suppression or addition of one or more nucleotides, and encoding        a peptide sequence derived from the aforementioned GBSSI of        Chlamydomonas reinhardtii, or derived from an aforementioned        peptide fragment of the latter, and having the property of        attaching to the starch granules, the said derived nucleotide        sequence preferably having a homology of at least about 50%, and        preferably of at least about 70%, with the aforementioned        nucleotide sequence or fragment,    -   or a nucleotide sequence capable of hybridizing with one of the        aforementioned nucleotide sequences or fragments, especially in        the strict conditions of hybridization defined above.

The invention relates more particularly to any recombinant nucleotidesequence as defined above, characterized in that the nucleotide sequencecoding for a peptide or polypeptide of interest is selected from thoseencoding biologically active peptides, especially peptides oftherapeutic interest or that can be used in the agricultural and foodindustry.

The invention also relates to any recombinant nucleotide sequence asdefined above, characterized in that the nucleotide sequence coding fora peptide or polypeptide of interest is selected from those encodingenzymes that are able to transform starch, such as the enzymes thatinteract with the α-glucans including various hydrolases,phosphorylases, α-1,4 glucanotransferases, branching enzymes, amylases,and especially the heat-resistant hydrolases obtained from extremophilessuch as the archaebacteria that are active at temperatures above 40° C.

The invention also relates to any recombinant nucleotide sequence asdefined above, characterized in that it comprises a nucleotide sequencecoding for a cleavage site, the said nucleotide sequence beingpositioned between the nucleotide sequence coding for a starch synthase,or a protein derived from the latter, and the nucleotide sequenceencoding the polypeptide of interest.

As an illustration, the nucleotide sequence coding for a cleavage siteis selected from the sequences coding for a peptide sequence of theaspartyl-proline type, which is very unstable at acid pH, or coding fora small peptide sequence recognized specifically by a protease, such astrypsin, chymotrypsin, pepsin, collagenase, thrombin, alasubtilisin, orrecognized by chemical compounds such as cyanogen bromide.

The invention also relates to any recombinant nucleotide sequence asdefined above, characterized in that it comprises a promoter locatedupstream of the nucleotide sequence coding for a starch synthase, or aprotein derived from the latter, as well as a sequence coding fortranscription termination signals located downstream of the nucleotidesequence encoding the polypeptide of interest.

Among the transcription promoters suitable for use within the scope ofthe present invention, we may mention:

-   -   for prokaryotic promoters, the Lac or T7 promoters,    -   for the eukaryotic promoters of higher plant type, the promoter        35S CaMV, or any type of promoter of plant origin,    -   in the case of the transformation of micro-algae, the promoter        used can be that of the ARG7 gene encoding arginosuccinate lyase        or the promoter of the NIT1 gene encoding nitrate reductase.

The invention also relates to any recombinant vector, especially of theplasmid, cosmid or phage type, characterized in that it contains arecombinant nucleotide sequence according to the invention as definedabove, inserted in a site that is non-essential for its replication.

The invention also relates to any cellular host, transformed by arecombinant vector as defined above, especially any bacterium such asAgrobacterium tumefaciens, and comprising at least one recombinantnucleotide sequence according to the invention.

The invention also relates to any fusion polypeptide characterized inthat it comprises:

-   -   in the N-terminal position, a starch synthase, or a protein        derived from that enzyme, especially by suppression, addition or        substitution of one or more amino acids, the said starch        synthase or derived protein having the property of migrating to        the sites of biosynthesis of the starch granules in plant cells        and of attaching to the starch granules,    -   and, in the C-terminal position, a peptide or polypeptide of        interest,    -   the C-terminal part of the amino acid sequence of the starch        synthase, or of the derived protein, being thus bound to the        N-terminal part of the peptide sequence of interest, the said        fusion polypeptide being encoded by a recombinant nucleotide        sequence as defined above according to the invention.

The invention relates more particularly to any fusion polypeptide asdefined above, characterized in that it includes, in the N-terminalposition, a GBSS that occurs in particular in plants, algae ormicro-algae, and more particularly an isoform GBSSI, or a proteinderived from the latter as defined above.

The invention relates more particularly to any fusion polypeptide asdefined above, characterized in that the starch synthase is selectedfrom:

-   -   the GBSSI of Chlamydomonas reinhardtii of about 640 to 680 amino        acids, of which the amino terminal end corresponds to the        following succession of amino acids: ALDIVMVAAEVAPGGKTGGLGDV        (SEQ ID NO:13), or ALDIVMVAAEVAPWSKTGGLGDV (SEQ ID NO:14), and        the carboxy terminal end corresponds to the succession of amino        acids shown in FIG. 1, the said GBSSI being encoded by the        nucleotide sequence obtained by screening a cDNA library        prepared from cells of Chlamydomonas reinhardtii, by means of an        antiserum obtained by immunization of rabbits with the starch        extracted from the aforementioned cells of Chlamydomonas        reinhardtii,    -   or a peptide fragment of the GBSSI of Chlamydomonas reinhardtii,        the said peptide fragment comprising the whole of the amino        terminal part of the said GBSSI, and being delimited at its        carboxy terminal end by the amino acid located in one of the        positions 25 to 238, or in one of the positions 118 to 238, of        the amino acid sequence shown in FIG. 1,    -   or a peptide sequence derived from an aforementioned peptide        sequence or fragment, especially by substitution, suppression or        addition of one or more amino acids, and having the property of        attaching to the starch granules, the said derived peptide        sequence preferably having a homology of at least about 60%, and        advantageously of at least about 80%, with the aforementioned        peptide sequence or fragment,    -   the property possessed by the GBSSI of Chlamydomonas        reinhardtii, or a fragment or a protein derived from the latter        as defined above, of being able to attach to the starch        granules, being measurable by the technique described above.

The invention relates more particularly to any fusion polypeptide asdefined above, characterized in that the starch synthase defined aboveis selected more particularly from:

-   -   the peptide sequence SEQ ID NO: 3 delimited by the amino acids        located at positions 1 to 708 in FIG. 2, corresponding to the        GBSSI of Chlamydomonas reinhardtii in the form of a pre-protein        of 708 amino acids,    -   any fragment as defined above from the peptide sequence SEQ ID        NO: 3 shown in FIG. 2, and more particularly any sequence of        which the amino acid of the amino terminal end corresponds to        that located in one of the positions 1 to 58 of SEQ ID NO: 3,        and of which the amino acid of the carboxy terminal end        corresponds to that located in one of the positions 495 to 708        of SEQ ID NO: 3, especially:        -   the sequence SEQ ID NO: 5 delimited by the amino acids            located at positions 58 to 708 of SEQ ID NO: 3,            corresponding to the GBSSI of Chlamydomonas reinhardtii in            the form of a mature protein of 651 amino acids,        -   the sequence SEQ ID NO: 7 delimited by the amino acids            located at positions 58 to 495 of SEQ ID NO: 3,            corresponding to a fragment of 438 amino acids of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,        -   the sequence SEQ ID NO: 9 delimited by the amino acids            located at positions 58 to 588 of SEQ ID NO: 3,            corresponding to a fragment of 531 amino acids of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,    -   or a peptide sequence derived from an aforementioned peptide        sequence or fragment, especially by substitution, suppression or        addition of one or more amino acids, and having the property of        attaching to the starch granules, the said derived peptide        sequence preferably having a homology of at least about 60%, and        advantageously of at least about 80%, with the aforementioned        peptide sequence or fragment,    -   the property possessed by the GBSSI of Chlamydomonas        reinhardtii, or a fragment or a protein derived from the latter        as defined above, of being able to attach to the starch        granules, being measurable by the technique described above.

The invention relates more particularly to any fusion polypeptide asdefined above, characterized in that the polypeptide of interest isselected from the biologically active peptides, especially the peptidesof therapeutic interest or that can be used in the agricultural and foodindustry.

The invention also relates to any fusion polypeptide as defined above,characterized in that the polypeptide of interest is selected from theenzymes that are able to transform starch, such as the enzymes thatinteract with the α-glucans, including various hydrolases,phosphorylases, α-1,4-glucanotransferases, branching enzymes, amylases,and especially the heat-resistant hydrolases obtained from extremophilessuch as the archaebacteria that are active at temperatures above 40° C.

The invention also relates to any fusion polypeptide as defined above,characterized in that it contains a cleavage site, as described above,positioned between on the one hand the starch synthase or a proteinderived from the latter, and on the other hand the polypeptide ofinterest.

The invention also relates to genetically transformed plant cells,containing one or more recombinant nucleotide sequences as describedabove, integrated in their genome or maintained in a stable manner intheir cytoplasm, the said plant cells being selected from the cells ofplants, algae or micro-algae, capable of producing starch.

The invention also relates to transgenic plant cells as described abovecontaining one or more fusion polypeptides defined above within thestarch granules contained in the said plant cells.

The invention relates more particularly to the aforementioned transgenicplant cells, transformed with a recombinant nucleotide sequencecontaining the nucleotide sequence of cDNA of about 2900 to 3100 basepairs, and of which the 1696 base pairs of the 3′ end are shown in FIG.1, the said nucleotide sequence coding for the GBSSI of Chlamydomonasreinhardtii described above, or containing a fragment or a derivedsequence as described above from the aforementioned cDNA.

The invention relates even more particularly to the aforementionedtransgenic plant cells, transformed with:

-   -   the cDNA nucleotide sequence shown in FIG. 2, the said        nucleotide sequence coding for the GBSSI of Chlamydomonas        reinhardtii,    -   any fragment as defined above of the nucleotide sequence shown        in FIG. 2,    -   or a derived nucleotide sequence, as defined above, from the        aforementioned nucleotide sequences,    -   or a nucleotide sequence capable of hybridization with one of        the aforementioned nucleotide sequences or fragments, especially        in the strict conditions of hybridization defined above.

The invention also relates to genetically transformed plants, algae ormicro-algae, or parts, especially flowers, fruits, leaves, stems, roots,seeds, or fragments of these plants, algae or micro-algae, containing atleast one recombinant nucleotide sequence as defined above integrated inthe genome or maintained in a stable manner in the cytoplasm of thecells of which they are composed.

The invention also relates to genetically transformed plants, algae ormicro-algae, or parts, or fragments of these plants, algae ormicro-algae, as defined above, containing one or more fusionpolypeptides as described above within the starch granules contained inthe plant cells of which they are composed.

Among the plants, algae or micro-algae transformed within the scope ofthe present invention, we may mainly mention wheat, maize, potato, rice,barley, amaranth, algae of the genus Chlamydomonas, especiallyChlamydomonas reinhardtii, algae of the genus Chlorella, especiallyChlorella vulgaris, or single-celled algae of the genus Dunaliella (asdescribed in the work “Dunaliella: Physiology, Biochemistry, andBiotechnology (1992), Mordhay Avron and Ami Ben-Amotz Publishers, CRCPress Inc., Boca Raton, Fla., USA”).

The invention relates more particularly to the aforementioned transgenicplants, algae or micro-algae, transformed with a recombinant nucleotidesequence containing the cDNA nucleotide sequence of about 2900 to 3100base pairs and of which the 1696 base pairs of the 3′ end are shown inFIG. 1, the said nucleotide sequence coding for the GBSSI ofChlamydomonas reinhardtii described above, or containing a fragment or aderived sequence such as are described above of the aforementioned cDNA.

The invention relates even more particularly to the aforementionedtransgenic plants, algae or micro-algae, transformed with:

-   -   the cDNA nucleotide sequence shown in FIG. 2, the said        nucleotide sequence coding for the GBSSI of Chlamydomonas        reinhardtii,    -   any fragment as defined above of the nucleotide sequence shown        in FIG. 2,    -   or a derived nucleotide sequence, as defined above, of the        aforementioned nucleotide sequences,    -   or a nucleotide sequence capable of hybridizing with one of the        aforementioned nucleotide sequences or fragments, especially in        the strict conditions of hybridization defined above.

The invention also relates to starch granules characterized in that theyinclude one or more fusion polypeptides defined above, the said starchgranules being further designated by the expression “transformed starchgranules” or “glucosomes”.

The invention relates more particularly to the aforementioned starchgranules comprising a fusion polypeptide defined above, the said fusionpolypeptide containing the GBSSI of Chiamydomonas reinhardtii of about640 to 680 amino acids described above, the amino terminal end of whichcorresponds to the following succession of amino acids:ALDIVMVAAEVAPGGKTGGLGDV (SEQ ID NO:113, or ALDIVMVAAEVAPWSKTGGLGDV (SEQID NO: 14), and the carboxy terminal end corresponds to the successionof amino acids shown in FIG. 1, or a fragment or a derived polypeptidesuch as are described above of the GBSSI of Chlamydomonas reinhardtii.

The invention relates more particularly to the aforementioned starchgranules comprising a fusion polypeptide defined above, the said fusionpolypeptide containing the sequence delimited by the amino acids locatedat positions 1 to 708 in FIG. 2 (SEQ ID NO:3), coding for the GBSSI ofChiamydomonas reinhardtii in the form of a pre-protein of 708 aminoacids, or any fragment as defined above of the peptide sequence shown inFIG. 2, especially any sequence in which the amino acid of the aminoterminal end corresponds to that located in one of the positions 1 to 58in FIG. 2 and SEQ ID NO:3, and in which the amino acid of the carboxyterminal end corresponds to that located in one of the positions 495 to708 in FIG. 2 and SEQ ID NO:3, such as the fragments mentioned above.

Advantageously, the aforementioned starch granules are characterized inthat they have a diameter between about 0.1 μm and several tens of μm,and in that the proportion by weight of the fusion polypeptides in thesegranules is between about 0.1% and 1%.

The invention also relates to any pharmaceutical compositioncharacterized in that it includes transformed starch granules as definedabove, if necessary in combination with a physiologically acceptablevehicle, the said granules containing one or more fusion polypeptides asdefined above, the peptide of interest in the said fusion polypeptidespossessing a defined therapeutic effect.

Advantageously the aforementioned pharmaceutical compositions of theinvention are in a form that can be administered parenterally,especially intravenously, or in a form that can be administered orally.

Preferably, the aforementioned pharmaceutical compositions that can beadministered parenterally are characterized in that the diameter of thestarch granules is between about 0.1 μm and several μm, especiallybetween about 0.1 μm and 10 μm, and in that the proportion by weight ofthe fusion polypeptides in these granules is between about 0.1% and 1%.

Starch granules as described above, with small diameters between about0.1 μm and about 10 μm, in which the proportion by weight of fusionpolypeptides is between about 0.1% and 1%, are obtained advantageously:

-   -   from plants or cells of plants transformed within the scope of        the present invention and selected for their property of        producing the aforementioned starch granules naturally, the said        plants being selected in particular from rice and amaranth,    -   or from parts of transformed plants within the scope of the        present invention, the said parts of these plants producing the        aforementioned starch granules naturally, such as the leaves of        the plants,    -   or from plants or cells of plants transformed within the scope        of the present invention, these plants being selected from        plants that have mutations such that they produce starch        granules of small diameters as mentioned above, especially from        the mutant plants described in Buléon A. et al., 1998,    -   or from plants or cells of plants transformed within the scope        of the present invention, these plants being selected from the        plants transformed with the aid of antisense nucleotide        sequences of all or part of the gene coding for ADP-glucose        pyrophosphorylase required for the synthesis of ADP-glucose in        plant cells, and especially from the transformed plants        described in the article by Müller-Röber B. et al., 1992.

Advantageously, in the case of pharmaceutical compositions mentionedabove that can be administered parenterally, the starch granules arepreferably selected from those of amorphous structure in the case whenwe wish to obtain rapid release of the fusion polypeptide that theycontain in the patient's blood, or conversely, from those of crystallinestructure when we wish to release the fusion polypeptide progressivelyin the blood.

By way of illustration, amorphous starch granules can be obtained fromseeds transformed according to the invention at the germination stage,or from specific mutant plants such as described by Shannon J. andGarwood D., 1984, especially from the mutant cultivars such as “amyloseextender” of maize or indeed all mutant cultivars of plants, algae ormicro-algae whose starch is amylose-enriched.

The starch granules according to the invention of crystalline structure,advantageously have about 30 to 35% of crystals, and can be obtainedfrom seeds of plants, especially of cereals, that have just beenharvested and at maturity, or from mutant plants such as described byShannon J. and Garwood D., 1984, especially from the mutant cultivarssuch as “waxy” of maize or indeed all the mutant cultivars of plants,algae or micro-algae whose starch is devoid of amylose.

The invention also relates to any pharmaceutical compositioncharacterized in that it includes one or more fusion polypeptides asdefined above, if necessary in combination with a physiologicallyacceptable vehicle, the peptide of interest in the said fusionpolypeptides possessing a defined therapeutic effect.

The invention also relates to any food composition as described above,characterized in that it contains transformed starch granules as definedabove, the said granules containing one or more fusion polypeptides asdefined above, the peptide of interest in the said fusion polypeptidesbeing usable in the food-processing field.

The invention also relates to any food composition as described above,characterized in that it contains one or more fusion polypeptides asdefined above, the peptide of interest in the said fusion polypeptidesbeing usable in the food-processing field.

The present invention also relates to any method of obtaining plantcells (from plants, algae or micro-algae), and, if necessary, from wholeplants, algae or micro-algae, transformed by at least one nucleotidesequence as defined above, characterized in that it comprises:

-   -   the transformation of plant cells, in such a way as to integrate        in the genome of these cells, or maintain in a stable manner in        their cytoplasm, one or more recombinant nucleotide sequences        according to the invention, and cultivation of these transformed        cells in vitro,    -   if necessary, the production of transformed plants from the        aforementioned transformed cells.

According to one embodiment of the aforementioned method of theinvention, the transformation of plant cells can be carried out bytransfer of the recombinant nucleotide sequence of the invention in theprotoplasts, especially after incubation of the latter in a solution ofpolyethylene glycol (PEG) in the presence of divalent cations (Ca²⁺)according to the method described in the article by Krens et al., 1982.

Transformation of the plant cells can also be carried out byelectroporation especially according to the method described in thearticle by Fromm et al., 1986.

Transformation of the plant cells can also be carried out using a genegun, by means of which metal particles coated with recombinantnucleotide sequences according to the invention are propelled at highvelocity, thus delivering genes to the interior of the cell nucleus,especially in accordance with the technique described in the article bySanford, 1988.

Another method of transformation of plant cells is the method ofcytoplasmic or nuclear micro-injection as described in the article by DeLa Penna et al., 1987.

According to a particularly preferred embodiment of the aforementionedmethod of the invention, the plant cells are transformed by putting thelatter in the presence of a cellular host transformed by a vectoraccording to the invention, as described above, the said cellular hostbeing able to infect the said plant cells making it possible tointegrate in the genome or maintain in a stable manner in the cytoplasmof the latter, recombinant nucleotide sequences of the inventioninitially contained in the genome of the aforementioned vector.

Advantageously, the aforementioned cellular host employed isAgrobacterium tumefaciens, especially according to the methods describedin the articles of Bevan, 1984 and of An et al., 1986, or Agrobacteriumrhizogenes, especially according to the method described in the articleby Jouanin et al., 1987.

Among the plant cells capable of being transformed within the scope ofthe present invention, we may mention mainly the cells of wheat, maize,potato, rice, barley, amaranth, Chlamydomonas reinhardtii, Chlorellavulgaris.

According to one embodiment of the aforementioned method of theinvention, the plant cells transformed according to the invention arecultivated in vitro, especially in bioreactors according to the methoddescribed in the article by Brodelius, 1988, in a liquid medium, oraccording to the method described in the article by Brodelius et al.,1979, in immobilized form, or according to the method described in thearticle by Deno et al., 1987, by culture of roots transformed in vitro.

According to a preferred embodiment of the aforementioned method of theinvention, the transformation of plant cells is followed by a stage ofobtaining transformed plants by culturing the said transformed cells ina suitable medium, and, if necessary, fertilization and recovery of theseeds of the plants obtained in the preceding stage, and cultivation ofthese seeds to obtain plants of the next generation.

The seeds transformed according to the invention are harvested from theaforementioned transformed plants, these plants being either those ofthe T0 generation, i.e. those obtained from culture of transformed cellsof the invention on a suitable medium, or advantageously those of thenext generations (T1, T2 etc.) obtained by self-fertilization of theplants of the preceding generation and in which the recombinantnucleotide sequences of the invention are reproduced in accordance withMendel's laws, or the laws of extrachromosomal inheritance.

The invention also relates to a method of preparation of transformedstarch granules as described above, characterized in that it comprises astage of extraction of the starch granules from transformed plant cellsor from plants, or from parts, especially flowers, fruits, leaves,stems, roots, or from fragments of these plants, transformed asmentioned above, especially by sedimentation in the conditions describedlater.

Preferably, the starch granules according to the invention are thoseobtained by extraction from transformed plants, algae or micro-algae,described above, or from parts, or fragments of these plants, algae ormicro-algae, defined above, especially by sedimentation in theconditions described later.

The transformed plants used for recovering the starch granules are thoseof the T0 generation, or advantageously those of the next generations(T1, T2 etc.) mentioned above.

The invention also relates to a method of preparation of fusionpolypeptides as defined above, characterized in that it comprises astage of recovery, and if necessary of purification, of the fusionpolypeptides from the aforementioned transformed starch granulesespecially in the conditions described later.

The invention also relates to a method of preparation of a peptide ofinterest, characterized in that it comprises the implementation of amethod as described above for obtaining plant cells or transformedplants according to the invention, the said method being carried out bytransformation of plant cells with the aforementioned nucleotidesequences coding for a fusion polypeptide containing a cleavage site asdescribed above, and includes an additional stage of cleavage of thesaid fusion polypeptide, by means of a suitable reagent, then, ifnecessary, a stage of purification of the polypeptide of interest.

The invention also relates to a method of biotransformation of starchgranules, characterized in that it comprises the following stages:

-   -   transformation of plant cells as defined above with the aid of        host cells described above containing one or more nucleotide        sequences coding for enzymes capable of transforming starch as        mentioned above,    -   production of plants, algae or micro-algae transformed in such a        way that their genome contains one or more nucleotide sequences        described above, by culture in vitro of the aforementioned        transformed plant cells,    -   if necessary, fertilization and recovery of the seeds of the        plants obtained in the preceding stage, and culture of these        seeds to obtain plants of the next generation,    -   extraction of starch granules from the aforementioned        transformed plants, algae or micro-algae, or from parts,        especially flowers, fruits, leaves, stems, roots, or from        fragments of these plants, algae or micro-algae, especially by        sedimentation in the conditions described later,    -   if necessary, heating of the said starch granules to a        temperature at which the peptide of interest of the        aforementioned fusion polypeptide is capable of being active.

Preferably, when the methods described above are carried out bytransformation of plant cells, the latter are transformed with theaforementioned recombinant sequences containing the cDNA nucleotidesequence of about 2900 to 3100 base pairs, and of which the 1696 basepairs of the 3′ end are shown in FIG. 1, the said nucleotide sequencecoding for the GBSSI of Chlamydomonas reinhardtii described above, andmore particularly with recombinant sequences as mentioned abovecontaining the nucleotide sequence shown in FIG. 2, or containing afragment or a derived sequence as described above of the nucleotidesequence shown in FIG. 2. The use of these recombinant sequencescontaining the nucleotide sequence coding for the GBSSI of Chlamydomonasreinhardtii described above makes it possible advantageously to avoidthe development of effects of co-suppression in the transformed plantsthus obtained.

The invention also relates to a method of preparation of antibodiesspecifically recognizing a starch synthase bound to the starch granule,of a given plant, algae or micro-algae, by immunization of an animal,especially of a rabbit, with the starch obtained from the said plant,algae or micro-algae.

Therefore, the invention relates more particularly to a method ofpreparation of antibodies specifically recognizing the GBSSI, of a givenplant, algae or micro-algae, by immunization of an animal, especially ofa rabbit, with the starch obtained from the said plant, algae ormicro-algae.

The invention also relates more particularly to a method of preparationof antibodies specifically recognizing an isoform of GBSS other thanGBSSI, from a given plant, algae or micro-algae, by immunization of ananimal, especially a rabbit, with the starch obtained from the saidplant, algae or micro-algae having a mutation such that the expressionof the GBSSI is suppressed, for example a mutation selected from thefollowing: sta2-29::ARG7 in Chlamydomonas reinhardtii (described byDelrue et al., 1992, mentioned above), amf in the potato (described byHovenkamp-Hermelink et al., 1987, mentioned above), wx in maize, riceand wheat (described by Tsai; 1974, mentioned above), lam in the pea(described by Denyer et al., 1995, mentioned above).

The invention also relates to a method of obtaining starch synthase,such as GBSS, and more particularly for the isoform GBSSI, from a givenplant, algae or micro-algae, by screening a cDNA library prepared fromcells of the said given plant, algae or micro-algae, capable ofcontaining this enzyme, using an antiserum containing antibodiesspecifically recognizing the said enzyme encoded by one or more cDNA'sfrom the library, when the said enzyme is expressed by a suitablecloning vector, the said antiserum being obtained according to themethod mentioned above.

The invention also relates to the nucleotide sequences coding for astarch synthase or for a derived protein, selected from:

-   -   the cDNA nucleotide sequence shown in FIG. 2, corresponding to        SEQ ID NO: 1 in the sequence list given later, the said        nucleotide sequence coding for the GBSSI of Chlamydomonas        reinhardtii,    -   any fragment as defined above of the nucleotide sequence SEQ ID        NO: 1 shown in FIG. 2, and more particularly any sequence whose        nucleotide of the 5′ end corresponds to that located in one of        the positions 1 to 186 of SEQ ID NO:1, and whose nucleotide of        the 3′ end corresponds to that located in one of the positions        1499 to 3117 of SEQ ID NO: 1, especially:        -   the sequence SEQ ID NO: 2 delimited by the nucleotides            located at positions 15 to 2138 of SEQ ID NO: 1, coding for            the GBSSI of Chlamydomonas reinhardtii in the form of            pre-protein of 708 amino acids (SEQ ID NO: 3) delimited by            the amino acids located at positions 1 and 708 of the            peptide sequence shown in FIG. 2,        -   the sequence SEQ ID NO: 4 delimited by the nucleotides            located at positions 186 to 2138 of SEQ ID NO:1, coding for            the GBSSI of Chlamydomonas reinhardtii in the form of mature            protein of 651 amino acids SEQ ID NO: 5) delimited by the            amino acids located at positions 58 and 708 of the peptide            sequence shown in FIG. 2,        -   the sequence SEQ ID NO: 6 delimited by the nucleotides            located at positions 186 to 1499 of SEQ ID NO: 1, coding for            a fragment of 438 amino acids (SEQ ID NO: 7) delimited by            the amino acids located at positions 58 and 495 of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,        -   the sequence SEQ ID NO: 8 delimited by the nucleotides            located at positions 186 to 1778 of SEQ ID NO: 1, coding for            a fragment of 531 amino acids (SEQ ID NO: 9) delimited by            the amino acids located at positions 58 and 588 of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,    -   or a nucleotide sequence derived by degeneration of the genetic        code of the aforementioned nucleotide sequences, and coding for        the aforementioned GBSSI of Chiamydomonas reinhardtii, or for an        aforementioned peptide fragment of the latter,    -   or a nucleotide sequence derived from an aforementioned        nucleotide sequence or fragment, especially by the substitution,        suppression or addition of one or more nucleotides, and encoding        a peptide sequence derived from the aforementioned GBSSI of        Chlamydomonas reinhardtii, or derived from an aforementioned        peptide fragment of the latter, and having the property of        attaching to the starch granules, the said derived nucleotide        sequence preferably having a homology of at least about 50%, and        preferably of at least about 70%, with the aforementioned        nucleotide sequence or fragment,    -   or a nucleotide sequence capable of hybridizing with one of the        aforementioned nucleotide sequences or fragments, especially in        the strict conditions of hybridization defined above.

The invention also relates to the polypeptides selected from:

-   -   the peptide sequence SEQ ID NO: 3 delimited by the amino acids        located at positions 1 to 708 in FIG. 2, corresponding to the        GBSSI of Chlamydomonas reinhardtii in the form of pre-protein of        708 amino acids,    -   any fragment as defined above of the peptide sequence SEQ ID NO:        3 shown in FIG. 2, and more particularly any sequence whose        amino acid of the amino terminal end corresponds to that located        in one of the positions 1 to 58 of SEQ ID NO: 3, and whose amino        acid of the carboxy terminal end corresponds to that located in        one of the positions 495 to 708 of SEQ ID NO: 3, especially:        -   the sequence SEQ ID NO: 5 delimited by the amino acids            located at positions 58 to 708 of SEQ ID NO: 3,            corresponding to the GBSSI of Chlamydomonas reinhardtii in            the form of mature protein of 651 amino acids,        -   the sequence SEQ ID NO: 7 delimited by the amino acids            located at positions 58 to 495 of SEQ ID NO: 3,            corresponding to a fragment of 438 amino acids of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,        -   the sequence SEQ ID NO: 9 delimited by the amino acids            located at positions 58 to 588 of SEQ ID NO: 3,            corresponding to a fragment of 531 amino acids of the            peptide sequence of the GBSSI of Chlamydomonas reinhardtii            shown in FIG. 2,    -   or a peptide sequence derived from an aforementioned sequence or        peptide fragment, especially by substitution, suppression or        addition of one or more amino acids, and having the property of        attaching to the starch granules, the said derived peptide        sequence preferably having a homology of at least about 60%,        advantageously at least about 80%, with the aforementioned        peptide sequence or fragment,    -   the property possessed by the GBSSI of Chlamydomonas        reinhardtii, or a fragment or a protein derived from the latter        as defined above, of being able to attach to the starch        granules, being measurable by the method described above.

The invention also relates to polyclonal or monoclonal antibodies,directed against the aforementioned polypeptides.

The invention will be further illustrated by means of the followingdetailed description of cloning of the gene coding for the GBSSI ofChlamydomonas reinhardtii, and obtaining transformed starch granulescontaining a fusion polypeptide with the said GBSSI, as well as by meansof FIG. 1 showing the nucleotide sequence and the protein sequencededuced from the cDNA insert of the CD142 clone coding for the GBSSI ofChlamydomonas reinhardtii (the underlined sequence corresponds to one ofthe three regions that are highly conserved across all the starch andglycogen synthases and is probably involved in fixation of theADP-glucose substrate).

I) Cloning of the cDNA (Complementary DNA) and gDNA (Genomic DNA)Sequences Corresponding to the Structural Gene of the GBSSI ofChlamydomonas reinhardtii.

A) Cloning of the cDNA

The strategy developed for cloning the cDNA corresponding to thestructural gene of the GBSSI of Chlamydomonas reinhardtii makes use ofscreening of an expression library using a polyclonal antiserum. Theantiserum is able to recognize a polypeptide sequence encoded by a cDNAexpressed from a suitable cloning vector.

a) Production of the Antiserum

In order to produce an antiserum capable of specifically recognizing theGBSSI of C. reinhardtii, the starch obtained from the wild strain (137C)was injected on three occasions into an albino New Zealand hybridrabbit. In a similar experiment, the residual starch from a doublemutant strain at loci STA2 and STA3 (IJ2) was injected in the rabbit inthe same conditions.

Detailed Protocols:

-   -   Genotypes of the strains of C. reinhardtii:    -   137C: mt-nit1 nit2    -   IJ2: mt-nit1 nit2 sta2-29::ARG7 sta3-1

The 137C strain is the reference strain for all the studies of starchmetabolism carried out in C. reinhardtii. The IJ2 strain was fullydescribed by Maddelein et al. in 1994. In this double mutant strain atthe STA2 and STA3 loci, the GBSSI and SSII activities are absentsimultaneously. The mutation at the STA2 locus was generated by geneinterruption by means of the pARG7 plasmid (Maddelein et al., 1994) andleads to complete disappearance of the GBSSI from the starch granule,whereas the mutant allele of the STA3 gene was generated by mutagenesisby X-rays (Fontaine et al., 1993).

Conditions for culture, extraction and purification of the starch: thecells were cultured for 3 days in the TAP medium with continuousillumination (3000 lux) from an inoculum of 5×10⁴ cells/ml. The mainculture is stopped when the cell concentration reaches about 2×10⁻⁶cells/ml.

Composition of the TAP medium (values for one liter of medium): NH₄Cl0.40 g ZnSO₄.7H₂O 22 mg Tris 2.40 g H₃BO₃ 11.4 mg KH₂PO₄ 0.32 gMnCl₂.4H₂O 5.1 mg K₂HPO₄ 1.47 g FeSO₄.7H₂O 4.2 mg CaCl₂.2H₂O 0.05 g MoO₃1.8 mg MgSO₄.7H₂O 0.30 g CoCl₂.6H₂O 1.6 mg EDTA 50 mg CuO₄.5H₂O 1.6 mgThe pH of the medium is adjusted to 7 with glacial acetic acid

The TAP-N medium has the same base composition, but this medium differsfrom the first by the absence of nitrogen supplied in the form ofammonium chloride, which is replaced with sodium chloride at the sameconcentration; it is in these culture conditions that the cellsaccumulate a quantity of starch representing up to twenty times that ofcells cultivated in TAP medium. In this case, culture is conducted for 5days in continuous light starting from a culture inoculated at 5×10⁻⁵cells/ml.

The cells are then concentrated by centrifugation at 2−4×10⁻⁸ cells/ml(Tris/acetate buffer pH 7.5 50 mM; EDTA 10 mM; DTT 2.5 mM) thensubjected to the action of a French press at 10000 psi. The extractobtained at press discharge is centrifuged at 5000 g for 15 min at 4° C.The deposit containing the starch is resuspended in one volume of water,to which are added nine volumes of Percoll (Pharmacia, Uppsala, Sweden)before being centrifuged at 10000 g for 30 min at 4° C. The Percollforms a density gradient during centrifugation. The starch, which has ahigh density (1.3 to 1.5), settles to the bottom of the tube whereas thelipids and other cell debris of low density form a “cap” at the surfaceof the Percoll gradient. The starch deposit is then rinsed three timeswith deionized water then stored at 4° C. after removing from it thelast supernatant from rinsing.

Conditions for immunization of the rabbit, taking and preparation of theantiserum: the rabbit used in this experiment is an albino New Zealandhybrid rabbit. Three successive injections were made at intervals ofthree weeks with 20 mg of purified starch suspended in 500 μl of water.500 μl of standard Freud adjuvant was added to this suspension. Bloodsamples were taken from the rabbit 3 weeks after the last injection. Theserum is prepared by the single centrifugation of the blood after 24hours of coagulation at 4° C. The antisera generated by the injectionsof the starches of the 137C and IJ2 strains are identified in thefollowing by the designations “antiserum SA137C” and “antiserum SAIJ2”respectively.

b) Preparation and Screening of the cDNA Library

The cDNA library was produced from mRNA's purified from the wild strainof C. reinhardtii. The λ ZAP expression vector was used.

Detailed Protocols:

Preparation of the complete RNA's of C. reinhardtii: this method is anadaptation of the method used for extracting RNA's from the leaves ofArabidopsis thaliana. The cells of a culture of 1−2×10⁻⁶ cells/ml areharvested by centrifugation at 3500 g for 15 min at 4° C. The cells arethen divided into aliquots with a volume of about 200 μl. At this stage,the cells are frozen in liquid nitrogen and can be stored at −80° C. forseveral months. 400 μl of “Z6” buffer of the following composition isadded to the 200 μl of frozen cells:

Buffer Z6: MES/NaOH pH 7.0  20 mM EDTA  20 mM Guanidine-HCl  6 Mβ-Mercaptoethanol 100 μM.

The mixture is stirred very vigorously for several minutes, then 400 μlof phenol/chloroform/isoamyl alcohol mixture (25 v/24 v/1 v) is addedand the mixture is stirred vigorously again for several minutes. Thewhole is centrifuged at 13000 g for 10 min at 4° C. After recovering andthen estimating the volume of the supernatant, 1/20 volume of aceticacid at 1 M as well as 0.7 volume of 100% ethanol are added. The nucleicacids are given time to precipitate at −20° C. for at least 30 min.After centrifugation at 13000 g for 15 min at 4° C., the pellet isresuspended in 400 μl of 3 M sodium acetate pH 5.6 then centrifuged for10 min at 13000 g at 4° C. The pellet is then rinsed twice with 70%ethanol, dried and finally dissolved in 50 μl of water treated withDEPC. The quantity of nucleic acids is determined in a spectrophotometerat 260 nm (OD₂₆₀=1 is equivalent to about 40 μg/ml of nucleic acids).

Construction of a cDNA library in the λ ZAP vector: the RNA's having apolyA tail (the mRNA's in particular) are isolated from the total RNApreparation using the kit “polyA Ttract mRNA isolation systems” marketedby Promega (Madison, Wis., USA). Synthesis of the cDNA's, ligation inthe λ ZAP vector and packaging in the capsids are effected using the kit“cDNA synthesis kit, ZAP-cDNA synthesis kit and ZAP-cDNA gigapack IIgold cloning kit” marketed by Stratagene (La Jolla, Calif., USA). Theprocedure followed corresponds to the instruction manual supplied withthe kit.

Immunological screening of a cDNA library in an expression vector:screening of the cDNA expression library of λ ZAP from C. reinhardtiiwas carried out using the antiserum previously obtained (see above).About 100000 lysis plates are spread by the Top-agar technique onseveral Petri dishes containing bacterial growth medium and the adaptedantibiotic. After incubation for 3 hours at 37° C., nitrocellulosefilters (Protan BA 85, Schleicher & Schuell, Dassel, Germany),previously immersed in a solution of IPTG 10 mM and dried, are appliedto the surface of the Top-agar. The dishes are incubated again for 3hours at 37° C. before being stored at 4° C. for 30 min. Thenitrocellulose filters are then carefully removed from the agar surface.The E. coli strain XL1-blue was used during screening of the λ ZAPlibrary. The protocol for filter development is then the same as thatused in the Western Blot study (see the section dealing with WesternBlot).

The positive lysis regions are subjected to two successive series ofscreening with the same antiserum in order to confirm their positivecharacter, and also to purify them. When a lysis region is found to bepositive at the end of three screenings, the sequence of the plasmidpBluescript SK+ containing the insert of interest is excised from the λphage in vivo. It is the “ExAssist helper phage” that is used forcotransfection of the SOLR strain with the λ ZAP phage. In this way weobtain a phagemid that is used for infecting the strain XL1-Blue MRF′leading to restoration of the double-stranded plasmid pBluescript SK+bearing the cDNA of interest.

Screening of this kind, conducted with the SA137C antiserum, led to theproduction of a single positive clone after three screenings. Wedesignated this clone “CD142”. The insert of the CD142 clone has a sizeof 1696 bp (see the sequence in FIG. 1).

c) Sequence Analysis of the Insert of the CD142 Clone

When the protein sequence libraries are interrogated with the sequencederived from the cDNA clone “CD142”, the greatest similarities areobtained with the GBSSI of the higher plants. This first indication ofthe origin of this cDNA is reinforced by the presence of an extension of119 amino acids (about 14 kDa) in the carboxy terminal position of thecoding sequence, relative to the main GBSSI's of the higher plants. Infact, the molecular weight of the GBSSI of C. reinhardtii, determined bySDS-PAGE, is on average 10 to 15 kDa higher than that of thecorresponding proteins in plants. The 119 amino acid extension mightexplain this difference in molecular weight between GBSSI's of differentorigins. Taken separately, this extension of the coding sequence doesnot share any similarity with other known types of polypeptidesequences.

The presence of the UAA stop codon in position 717 signals the start ofa very long non-coding region of 946 bp. These noncoding regions in 3′terminal position, which frequently occur in the nuclear genes ofChiamydomonas, seem in particular to be intended to stabilize themessenger.

B) Cloning of gDNA

The gDNA relating to the CD142 clone was isolated after screening anindexed gDNA library in cosmids (Zhang et al., 1994). Constructed in acosmid vector derived from c2RB, this gDNA library is contained in 12096-well microtitration plates. Each well (apart from two, to facilitateorientation of the plate) contains a bacterial clone transporting asingle cosmid. The whole library thus represents 11280 clones for whichthe average size of the inserts is approx. 38 kb. The nuclear genome ofC. reinhardtii is therefore represented there statistically about fourtimes.

Screening of this library with a probe corresponding to the CD142 cloneled to the isolation of a genomic DNA clone designated 18B1. The insertpresent in this single cosmid was analysed in more detail. Afterrestriction by NotI then hybridization with the CD142 probe, only a bandof about 9 kb remains positive, indicating that all the informationcorresponding to the CD142 clone is present in this fragment. Thegenomic sequence corresponding to the CD142 clone is presented below.

Detailed Protocols:

Preparation of nylon filters for screening: the nylon filters (Hybond N,Amersham Buchler, Braunschweig, Germany) are carefully placed on a Petridish containing a rich bacterial growth medium supplemented with theappropriate antibiotic (in the present case, ampicillin is used). EachE. coli clone contained in the library is then replicated directly onthe nylon filter using a replicating apparatus and the dishes thusprepared are incubated overnight at 37° C. The filters are then removedfrom the agar surface and subjected to the following treatment:

-   -   (1) 2 min with a denaturing solution (NaOH 0.5 M; NaCl 1.5 M)    -   (2) 2 min with a neutralizing solution (Tris/HCl pH 7.0 0.5 M;        NaCl 1.5 M)    -   (3) 2 min with a rinsing solution (buffer 2×SSC)

Finally the filters are incubated in a drying cabinet for 2 hours at 80°C.

Prehybridization and hybridization of the filters: prehybridization iscarried out in the hybridization buffer at 42° C. for at least 4 hours.Hybridization is effected at 42° C. for a whole night in the presence ofthe ³²P-labelled nucleotide probe. The membrane is washed at 60° C. inthe washing solution, adjusted to the stringency that we wish to apply.The time and frequency of replacement of the washing baths varydepending on the stringency and the radioactivity levels detected on themembrane. In general, the baths are renewed every 10 min and washingbegins with a washing buffer of low stringency and ends with a buffer ofgreater stringency. A Kodak X-OMAT AR film is finally exposed to thefilters at −80° C. in order to detect the positive clones.

Composition of the Solutions and Buffers:

Buffer SSC×20: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in800 ml of water. Adjust the pH to 7.0 with a few drops of a 10 Nsolution of NaOH. Make up to 1 liter with water.

Hybridization buffer: Formamide 50% Denhardt's ×5 SDS 0.5% Na phosphatebuffer pH 7.0 50 mM DNA of salmon sperm 100 μg/ml Bovine serum albumin0.5% Denhardt's reagent × 100 (quantity for 500 ml in water): Ficoll 40010 g PolyVinylPyrrolidone 40 (PVP40) 10 g BSA 10 g Phosphate buffer pH7.0 at 1 M (quantity for 100 ml of buffer): Na₂HPO₄ 1 M 57.7 ml NaH₂PO₄1 M 42.3 ml Washing buffers: low stringency: SSC × 2; SDS 0.2% mediumstringency: SSC × 1; SDS 0.5% high stringency: SSC × 0.5; SDS 0.5% SSC ×0.1; SDS 0.5%

Preparation and labelling of a nucleotide probe with ³²P: the fragmentserving as nucleotide probe is generally inserted in the multiplecloning site of a bacterial plasmid. It is therefore first necessary todigest it with the appropriate restriction endonucleases then separatethe fragment of interest from the rest of the plasmid by electrophoresison 1% agarose gel buffered with TAE buffer×1. The band corresponding tothe fragment of interest is then cut out of the gel and DNA extractionis effected with the kit “The GENECLEAN II Kit” marketed by BIO 101 Inc.(La Jolla, Calif., USA). The piece of agarose is firstly dissolved in a6 M sodium iodide solution. On completion of solution, the DNA moleculesare then captured with a silica matrix designated “Glassmilk”. The DNAmolecules, in the presence of the NaI chaotropic agent, are adsorbedspecifically on the silica beads. After eliminating the salts and thedissolved agarose, the DNA molecules are eluted from the silica beads inthe presence of sterile water.

Labelling of a nucleotide probe with ³²P is accomplished using the“Random primed DNA labelling kit” from Boehringer (Mannheim, Germany).The principle is random priming of the elongation reaction by Klenow DNApolymerase using a mixture of hexanucleotides representing all thepossible combinations of sequences. The radioactive element isincorporated starting from [α-³²P]-dCTP (3000 Ci/mmol) of which 50 μCiis used for each labelling reaction. The radiolabelled probe is finallyadded to the hybridization solution after denaturing at 95° C. for 4min.

C) The STA2 Locus in C. reinhardtii Represents the Structural Gene ofthe GBSSI

The following analysis demonstrates formally that the STA2 gene of C.reinhardtii corresponds to the structural gene of the GBSSI and that theCD142 clone represents a cDNA that comes from this locus. In fact,restriction analyses of the genomic DNA digested by the BamHIendonuclease reveal a profound change of the restriction profile in themutant BAFR1 strain at the STA2 locus generated by gene interruption(Delrue et al., 1992). The same change is also observed in the doublemutant IJ2 strain at the STA2 and STA3 loci which Maddelein et al.(1994) generated by crossing the BAFR1 strain with a sta3-1 mutantstrain.

Moreover, this change of the restriction profile in the meiotic progenyof the IJ2 strain fused with the “CS9” strain of C. smithii could befollowed in the following crossing: $\begin{matrix}{CS9} & X & {IJ2} \\\left( {C.\mspace{14mu}{smithii}} \right) & \; & \left( {C.\mspace{14mu}{reinhardtii}} \right) \\{+ {/ +}} & \; & {{sta2}\text{-}\text{2}{{9{::}{ARG7}}/{sta3}}\text{-}1} \\\; & \downarrow & \; \\\; & {+ {/ +}} & {{25\%}\mspace{110mu}} \\\; & {{sta2}\text{-}{{29{::}{ARG7}}/{sta3}}\text{-}1} & {25\%\mspace{25mu}{progeny}} \\\; & {{sta2}\text{-}{{29{::}{ARG7}}/ +}} & {25\%\mspace{31mu}{meiotic}} \\\; & {{/{sta3}}\text{-}1} & {{25\%}\mspace{104mu}}\end{matrix}$

352 segregants resulting from this crossing were purified, amplified andtheir starch accumulation phenotype was analysed. 54 meioticrecombinants underwent restriction analysis: 21 of genotypesta2-29::ARG7/+, 19 of genotype +/sta3-1 and 14 wild. With regard to the21 segregants of genotype sta2-29::ARG7/+their restriction profile,obtained by digestion with BamHI and hybridized with the CD142 probe,still has the same change as the parental strain IJ2. We deduce fromthis that the STA2 gene and the CD142 probe are very strongly linkedgenetically. There is no longer any doubt as to the nature of the CD142clone, which represents the structural gene of the GBSSI (the STA2locus).

Detailed Protocols:

Carrying out the crossings: before carrying out the fusion of cells withopposite sexual polarities, it is necessary to put them in a state thatis favourable to their fusion. Thus, the cells must first bedifferentiated into gametes before they are put in direct contact.Gametogenesis is induced in Chlamydomonas by subjecting the cells to anitrogen deficit and in the presence of a strong light source (5000lux). For this, fresh cells cultivated on rich agar medium (culture ofless than 5 days) are suspended in 2 ml of TAP-N medium and left for atleast 12 hours in strong light without agitation. The state of the cellsis examined with an optical microscope before being brought intocontact. After differentiation into gametes, the cells are smaller andin particular much more active than in the case of a non-deficientculture. Equivalent quantities of cells of each sexual polarity aremixed. Fusion is always carried out in strong light. After one hour ofcontact, cell fusions are already visible in the optical microscope.Analysis of the meiotic segregants will consist of depositing theproducts of cellular fusion on a rich medium at 4% agar. The dishes thusobtained are incubated in diffuse light for 15 hours then stored intotal darkness for at least a week. This permits maturation of thezygotes and their “encystment” in the 4% agar. After this period ofincubation in darkness, the dishes are returned to the light and thefollowing stages are carried out as quickly as possible. In order toeliminate the greatest possible number of unfused vegetative cells, thesurface of the agar is scraped very lightly with a razor blade.Observing with a binocular magnifier, a region containing about fiftyzygotes is marked off and these are transferred to a fresh dish of richmedium at 1.5% agar. To be sure of complete disappearance of residualvegetative cells, the dish is subjected to chloroform vapours for 45seconds to 1 minute (in contrast to other cells, zygotes can withstandthis moderate time of exposure to chloroform vapours). The presence oflight will irreversibly trigger the start of meiosis of the zygotes.During their germination (facilitated by the higher moisture content ofthe medium containing 1.5% agar) the zygotes will release four haploiddaughter cells (a tetrad), which will grow by mitotic divisions and formcolonies in the dish. Analysis of the meiosis products can be effectedin two ways. The first consists of random investigation of at least 200segregants resulting from the crossing. After purification of thesegregants, the characters of the latter can be studied by replicationon different selective media.

Techniques of extraction of genomic DNA: the protocol adopted forextraction of total DNA is that described by Rochaix et al. (1991); hereare the details:

-   -   (1) Centrifuge 10 ml of cell culture to about 3−5×10⁶ cells/ml        for 10 min at 3500 g in a 15 ml bottle.    -   (2) The pellet of cells is then resuspended in 350 μl of the        following buffer:

Tris/HCl pH 8.0  20 mM EDTA  50 mM NaCl 100 mM.

-   -   (3) Add 50 μl of proteinase K from a stock solution at 2 mg/ml        (if unavailable, it is possible to use pronase at 10 mg/ml).    -   (4) Add 25 μl of SDS at 20% and incubate for 2 hours at 55° C.    -   (5) Add 2 μl of diethylpyrocarbonate (DEPC) and incubate for 15        min at 70° C.    -   (6) Cool the tube briefly in ice and add 50 μl of 5 M solution        of potassium acetate.    -   (7) Mix, by shaking the tube correctly, and leave on ice for at        least 30 min (it is possible to stop the extraction at that        moment and resume on the next day if the tubes are left in ice        in a coldroom).    -   (8) Transfer to a 1.5 ml Eppendorf tube and centrifuge for 15        min in a minicentrifuge (at about 13000 g).    -   (9) Recover the supernatant, transferring it to a new Eppendorf        tube.    -   (10) Extract the supernatant with one volume of the following        mixture:

Phenol (saturated with TE: Tris/HCl pH 8.0 10 mM, EDTA 25 vol 1 mM)Chloroform 24 vol Isoamyl alcohol  1 vol

-   -   (11) After extraction, add 1 ml of 100% ethanol at room        temperature. A precipitate should be seen to appear in the form        of “angel hair” if extraction is successful. From this moment,        manipulations must be careful and gentle so that the DNA        molecules do not break.    -   (12) Centrifuge for 5 min in a minicentrifuge (about 13000 g).    -   (13) Rinse the pellet with 70% ethanol and centrifuge for 3 min        in a minicentrifuge.    -   (14) Repeat operation (13) once or twice for proper elimination        of the salts.    -   (15) Dry the pellet for 5 min at 37° C., then dissolve it in 50        μl of TE containing bovine pancreas RNase at 1 μg/ml.

Molecular hybridizations and Southern Blot analyses: 25 μg of DNA isdigested completely with the appropriate restriction endonuclease(s).The restriction products are then separated by electrophoresis in 0.8%agarose gel, TBE×1. Then the gel is incubated successively for 15 min inthe depurination solution and for 30 min in the denaturing solution. Thedenatured DNA is then transferred onto “Porablot NYplus” nylon membrane(Macherey-Nagel GmbH, Düiren, Germany) by capillarity with SSCbuffer×20. After transfer, the membrane is incubated at 80° C. inabsence of air for 2 hours to fix the DNA fragments to the surface ofthe nylon membrane. Prehybridization is effected in the hybridizationbuffer at 42° C. for at least 4 hours. Hybridization is accomplished at42° C. for a whole night in the presence of the labelled probe preparedpreviously. The membrane is washed at 60° C. in the washing bufferadjusted to the stringency that is to be applied to the washing. Thetime and frequency of replacement of the washing baths vary depending onthe stringency and the levels of radioactivity present on the membrane.In general, the baths are renewed every 10 min and washing begins with awashing buffer of low stringency and ends with a buffer of higherstringency. A Kodak X-OMAT AR film is finally exposed to the membrane at−80° C. to reveal the hybridization zones.

II) Investigation of Binding of the GBSSI to the Starch Granule.

A) Analysis of the sta2-1 Mutant Allele

Among all the mutant alleles generated at the STA2 locus in C.reinhardtii, just one leads to the production of a 58 kDa truncatedGBSSI in place of the 76 kDa wild protein. This is the sta2-1 allele ofthe 18B strain. Delrue et al. (1992), by micro-sequencing of the GBSSIextracted from a polyacrylamide gel, were able to demonstrate that theamino terminal peptide sequences of the proteins of the wild strain(137C) and of the mutant strain (18B) are identical.

-   -   Amino terminal sequences:    -   ρ GBSSI of the 137C strain: ALDIVMVAAEVAPGGKTGGLGDV (SEQ ID        NO:13)    -   ρ GBSSI of the 18B strain: ALDIVMVAAEVAPGGKTGGLGDV (SEQ ID        NO:13)

The protein produced by the sta2-1 mutant allele is therefore truncatedin the carboxy terminal position and the K_(m) for ADP-glucose isincreased by a factor of 6. Absence of this carboxy terminal sequencedoes not, however, alter the properties of fixation of the protein onthe granule, as is shown in FIG. 1.

Detailed Protocol:

Technique of extraction of the proteins from the starch granule andSDS-PAGE: the proteins are extracted from 0.3 to 1 mg of starch with 60μl of extraction buffer: β-mercaptoethanol 5% (v/v); SDS 2% (w/v) at100° C. for 5 min. After centrifugation at 13000 g for 10 min, thesupernatant is recovered and the operation is repeated once with thepellet. The two supernatants are combined and the sample can be loadedinto the gel wells after adding again 10 μl of the following loadingbuffer: Tris 50 mM, glycine 384 mM, 20% glycerol, SDS 0.1%, bromophenolblue 0.001%. Migration is carried out at room temperature, at 150 V for1 h 30 (until the bromophenol blue leaves the gel). The proteins arerevealed by staining with Coomassie blue or by immunodetection (seebelow; section relating to Western Blot). During staining with Coomassieblue, the gel is incubated for 30 min in the following solution: 2 g ofCoomassie Brilliant Blue R250, 0.5 g of Coomassie Brilliant Blue G250,425 ml of ethanol, 50 ml of methanol, 100 ml of acetic acid; watersufficient for 1000 ml. The gel is then decolorized using the followingsolutions:

-   -   15 to 30 min in decolorizer I: 450 ml of ethanol, 50 ml of        acetic acid; make up to 1000 ml with water.    -   one night in decolorizer II: 80 ml of acetic acid, 50 ml of        methanol, make up to 1000 ml with water; this decolorizer II        removes the nonspecific coloration of the gel.    -   decolorizer III (240 ml of acetic acid, 200 ml of methanol, make        up to 1000 ml with water) permits complete decolorizing of the        gel if necessary.

B) Determination of the Quantity of Proteins Bound to the Granule

The quantity of proteins bound to the starch granule was determined indifferent culture conditions and in various gene libraries. For this,the cells were placed in conditions of massive accumulation of starch(nitrogen-deficient medium) or in conditions of mixotrophic growth(presence of nitrogen). The proteins extracted from the granule werethen deposited on polyacrylamide gel in denaturing conditions (presenceof SDS). After migration, the proteins are revealed by staining withCoomassie blue. The 17 strain, mutant at the STA1 locus, was used duringthis experiment. This mutation was described in detail by Van denKoornhuyse et al. (1996) and then by Van de Wal et al. (1998). The STA1locus corresponds to the structural gene of the large regulatory subunitof ADP-glucose pyrophosphorylase. The sta1-1 mutation produced duringX-ray mutagenesis leads to insensitivity of the enzyme to3-phosphoglyceric acid, its allosteric activator. Consequently, the 17strain accumulates less than 5% of the normal quantity of starch. Theestimate of the quantity of GBSSI bound to the granule is approx. 0.1%of the weight of starch in conditions of nitrogen deficiency for the137C and 18B strains. This value reaches 1% in conditions of mixotrophy.In the case of the 17 strain, regardless of the culture conditions, theGBSSI represents more than 1% of the weight of the starch granule. Thetechniques employed in this analysis are the same as those described inthe preceding paragraph.

C) Analysis of Immune Response in Western Blot

To test the antigenicity of the SA137C and SAIJ2 antisera obtainedpreviously in the rabbit, the proteins extracted from 100 μg of freshstarch obtained from different strains cultivated in variable cultureconditions were subjected to analysis by the immunotransfer technique(Western Blotting). The immune response produced with respect to GBSSIduring injection of the starch from the wild strain (137C) proves veryspecific and strong (the proteins having been extracted from just 100 μgof fresh starch) even in the case of the truncated protein in the sta2-1mutant. The quantity of proteins bound to the starch granule seemslarger in the 17 mutant during nitrogen-deficient culture, as shown bythe presence of a mass band higher than GBSSI revealed by the SA137Cantiserum. This is confirmed by Western Blot analysis effected with theSAIJ2 antiserum, where the strongest immune response is detected withthe proteins extracted from the starch of the 17 strain cultivated withnitrogen deficiency.

For control purposes, we carried out the same type of experiment usingthe PA55 antiserum obtained by Abel et al. (1995). This antiserumproduced in the rabbit is directed against a peptide whose consensussequence corresponds to the strongly conserved carboxy terminal regionin all the starch synthases of higher plants, whether they are solubleor bound to the starch granule. This antiserum recognizes the GBSSI ofC. reinhardtii specifically when the latter is present in the granule.Moreover, the PA55 antiserum also recognizes the truncated proteinproduced by the 18B mutant (sta2-1). It therefore appears that thehighly conserved sequence in carboxy terminal position is still presentin the truncated protein.

Detailed Protocols:

Technique of protein extraction from the starch granule and SDS-PAGE:these techniques are the same as those described in the precedingsection apart from staining with Coomassie blue, which is omitted inthis case.

Technique of transfer and detection with antisera: when migration onSDS-PAGE has ended, the gel is incubated for 30 min in the “Western”buffer×1 containing 20% of methanol. The proteins are thenelectrotransferred onto a nitrocellulose membrane (Protan BA 85Schleicher & Schuell, Dassel, Germany) using electrotransfer apparatus(Multiphor II, LKB-Pharmacia, Bromma, Sweden) at 4° C. in the followingconditions: 45 min at 250 mA with the buffer used previously. After thisstage of transfer of the proteins onto the nitrocellulose membrane, thelatter is incubated for 1 hour at room temperature in TBST buffercontaining 3% of BSA. The membrane is then rinsed three times in TBSTbuffer before being incubated overnight at 4° C. in the rabbit primaryantiserum diluted in TBS buffer. The membrane is again rinsed threetimes with TBST buffer and is then incubated for 1 hour at roomtemperature with the biotinylated secondary antibody diluted at 1/500 inthe TBS directed against the rabbit antiserum. After three furtherrinses in TBST buffer, the membrane is incubated for 30 min at roomtemperature with the streptavidin-alkaline phosphatase complex at 1/3000dilution in the TBS buffer. Finally, after 3 rinses in TBST buffer, themembranes are developed by incubation in a diethanolamine buffercontaining the substrates of alkaline phosphatase: NBT and BCIP (theincubation time varies depending on the intensity of reaction). Thedetection kit used is the one offered by Amersham Buehler (Braunschweig,Germany): “Blotting detection kit for rabbit antibodies”

Compositions of solutions and buffers: “Western” buffer × 10: Glycine390 mM Tris 480 mM SDS  0.375% TBS buffer (Tris Buffer Saline) Tris/HClpH 7.5  20 mM NaCl 500 mM TBST buffer (Tris Buffer Saline Tween): TBS +0.1% (v/v) Tween 20 NBT: Nitro-Blue Tetrazolium in solution indimethylformamide 70% BCIP: 5-Bromo-4-Chloro-3-Indolyl Phosphate insolution in dimethylformamide. @

III) Targeting of Fusion Proteins in the Starch Granule

The specific carboxy terminal extension of the GBSSI of C. reinhardtiiis not required for targeting the protein to the starch granule in vivoas we were able to demonstrate in the previous experiments. Theextension of about 16 kDa can be replaced by a peptide sequence ofinterest, thus permitting its targeting to the very heart of the starchgranule.

The various types of vectors that can be constructed for applying thismethod in higher plants consist of:

-   -   a bacterial selector gene and a bacterial replication origin in        order to be able to amplify the plasmid in a suitable bacterial        strain    -   a selector gene that will permit easy selection of transformant        plants    -   translational fusion between the coding sequence of the GBSSI        and a polypeptide sequence of interest. Two main types of        translational fusions may be considered: in the first case, it        is the 58 kDa truncated sequence from GBSSI that is fused with        the sequence of interest; in the second case, the complete        sequence of the GBSSI is employed.    -   fusion can be put under the control of a strong constitutive        plant promoter, or of an inducible plant promoter, immediately        followed by a suitable transit peptide promoting translocation        of the fusion protein to the chloroplast.

IV) Protocol for Determination of the Activity of Granule-Bound StarchSynthase:

Add 20 μg of starch to 100 μl of the following reaction mixture:

Glycylglycine/NaOH pH 9.0 50 mM (NH₄)₂SO₄ 100 mM β-mercaptoethanol 5 mMMgCl₂ 5 mM Bovine serum albumin 0.5 mg/ml ADP-glucose 0.2 mM [U⁴C]ADP-glucose (235 mCi/mmol) 2.66 μg Trisodium citrate 0 or 0.5 M(specified depending on circumstances)

The reaction is carried out at 30° C. for 15 min and is then stopped byadding 3 ml of 70% ethanol. The precipitate obtained is filtered undervacuum on a “Whatman Glass Fibre” filter (Whatman, Maidstone, UK), andrinsed with 4×5 ml of 70% ethanol. A Beckman counter is used forradioactivity counting after the filters have been placed in countingphials containing 3.5 ml of scintillation liquid.

V) The methods of Starch Extraction and Purification are as Follows:

-   -   in the case of a single-celled green algae such as Chlamydomonas        reinhardtii (see the method described above)    -   in the case of seeds, tubers or any other organ of higher        plants:        the organ or the type taken from the plant is properly        homogenized (after grinding). The pulverized material thus        obtained is rinsed with water through a filter cloth (such as        Miracloth Calbiochem, La Jolla, Calif., USA). The filtrate is        then left to stand for two hours for the starch granules to        settle. The sediment is rinsed firstly with several volumes of        water then a second time with several volumes of NaCl solution        at 0.1 M. The sediment is filtered once again then rinsed twice        with ethanol before being dried.

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1. A pharmaceutical composition, comprising starch granules containingat least one fusion polypeptide comprising: In the N terminal position:the peptide sequence of SEQ ID No: 3 comprising the granule bound starchsynthase GBSSI of Chlamydomonas reinhardtii or the sequence SEQ ID No: 5comprising the GBSSI of Chlamydomonas reinhardtii in the form of matureprotein of 651 amino acids, said sequences being encoded by nucleotidesequences SEQ ID No: 2, and 4 respectively, and, in the C-terminalposition, a peptide or polypeptide of interest, the C-terminal part ofthe amino acid sequence of the GBSSI thus being bound to the N-terminalpart of the peptide sequence of interest, the said fusion polypeptidebeing encoded by a recombinant nucleotide sequence containing in the5′→3′ direction, a nucleotide sequence coding for said Chlamydomonasreinhardtii GBSSI, the said nucleotide sequence coding for this enzymebeing positioned upstream of a nucleotide sequence coding for thepeptide or polypeptide of interest, the peptide of interest in the saidfusion polypeptides possessing a defined therapeutic effect.
 2. Apharmaceutical composition according to claim 1, containing the sequenceSEQ ID No: 7 or the sequence SEQ ID No: 9, said sequence being encodedby nucleotide sequences SEQ ID Nos: 6 and 8, respectively.
 3. Apharmaceutical composition according to claim 1 wherein the peptide orpolypeptide of interest is selected from: biologically active peptides,or enzymes that are able to transform starch, such as enzymes thatinteract with α-glucans including various hydrolases, phosphorylases,α-1,4-glucanotransferases, branching enzymes, amylases.
 4. Apharmaceutical composition according to claim 1 wherein the fusionpolypeptide comprises a cleavage site positioned between the starchsynthase, and the polypeptide of interest.
 5. A pharmaceuticalcomposition according to claim 1, wherein the diameter of the starchgranules are between about 0.1 μm and of 10 μm, and the proportion byweight of the fusion polypeptides in these granules are between about0.1% and 1%.
 6. A pharmaceutical composition containing at least onefusion polypeptide comprising: in the N-terminal position: the peptidesequence SEQ ID No: 3 or the sequence SEQ ID No: 5 said sequence beingencoded by nucleotide sequences SEQ ID Nos: 2 and 4, respectively, and,in the C-terminal position, a peptide or polypeptide of interest, theC-terminal part of the amino acid sequence of the GBSSI thus being boundto the N-terminal part of the peptide sequence of interest, the saidfusion polypeptide being encoded by a recombinant nucleotide sequencecontaining in the 5′→3′ direction, a nucleotide sequence coding for saidChlamydomonas reinhardtii GBSSI, the said nucleotide sequence coding forthis enzyme being positioned upstream of a nucleotide sequence codingfor a peptide or polypeptide of interest, the peptide of interest in thesaid fusion polypeptides possessing a defined therapeutic effect.
 7. Apharmaceutical composition according to claim 6 containing the sequenceSEQ ID No: 7 the sequence SEQ ID No: 9 said sequences being encoded bynucleotide sequences SEQ ID Nos: 6 and 8 respectively.
 8. Apharmaceutical composition according to claim 6 wherein the peptide orpolypeptide of interest is selected from: biologically active peptides,or enzymes that are able to transform starch, such as enzymes thatinteract with α-glucans including various hydrolases, phosphorylases,α-1,4-glucanotransferases, branching enzymes, amylases.
 9. Apharmaceutical composition according to claim 6 wherein the fusionpolypeptide comprises a cleavage site positioned between the starchsynthase, and the polypeptide of interest.
 10. A pharmaceuticalcomposition according to claim 3, wherein the biologically activepeptides are peptides of therapeutic interest or peptides that can beused in the agricultural and food industry.
 11. A pharmaceuticalcomposition according to claim 3, wherein the enzymes that are able totransform starch are heat-resistant hydrolases obtained fromextremophiles such as archaebacteria that are active at temperaturesabove 12° C.
 12. A pharmaceutical composition according to claim 8,wherein the biologically active peptides are peptides of therapeuticinterest or peptides that can be used in the agricultural and foodindustry.
 13. A pharmaceutical composition according to claim 8, whereinthe enzymes that are able to transform starch are heat-resistanthydrolases obtained from extremophiles such as archebacteria that areactive at temperatures above 40° C.